發(fā)布時(shí)間：2018-05-09 16:24

  本文選題：日本血吸蟲 + SjGST蛋白��； 參考：《安徽醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的獲取高純度的日本血吸蟲重組GST蛋白( rSjGST) ,并制備出該蛋白的單克隆抗體,并對(duì)其相關(guān)的生物學(xué)特性進(jìn)行鑒定,建立日本血吸蟲病患者GST蛋白水平檢測(cè)方法。方法將含有重組質(zhì)粒pET28a-SjGST的工程菌BL21進(jìn)行培養(yǎng)并進(jìn)行IPTG誘導(dǎo)表達(dá)后,破碎大腸桿菌菌體并利用His-tag親和層析和高效液相色譜(HPLC)純化出rSjGST蛋白,純化出的目的蛋白使用十二烷基磺酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)以及免疫印跡技術(shù)進(jìn)行鑒定。之后采用B淋巴細(xì)胞雜交瘤技術(shù),以高純度的rSjGST蛋白免疫Balb/c雌性小鼠,常規(guī)聚乙二醇介導(dǎo)融合,以間接ELISA方法篩選陽性克隆,有限稀釋法亞克隆篩選單克隆細(xì)胞株,并使用秋水仙素對(duì)其核型進(jìn)行鑒定,使用ISO-2鼠源單克隆抗體亞型檢測(cè)試劑盒檢測(cè)其亞類,并檢測(cè)其效價(jià)、特異性等。建立以雙抗夾心法和間接ELISA法檢測(cè)血清GST水平的方法,并探討該方法的精密度、準(zhǔn)確性和特異性等。結(jié)果純化出大量的高純度rSjGST蛋白,并且篩選出能夠穩(wěn)定分泌抗rSjGST單抗的雜交瘤細(xì)胞株1B11和3C12。用試劑盒檢測(cè)出此單抗的亞型為IgG2b和IgG1。結(jié)論依靠大腸桿菌表達(dá)系統(tǒng),本實(shí)驗(yàn)高效表達(dá)出了rSjGST蛋白,并以此rSjGST蛋白制備出了單克隆抗體1B11和3C12,所建立的雙抗體夾心ELISA檢測(cè)血清rSjGST蛋白水平的方法為今后的血吸蟲病免疫診斷提供了研究基礎(chǔ),這項(xiàng)診斷技術(shù)同樣可以應(yīng)用于臨床日本血吸蟲病的血清檢測(cè)。
[Abstract]:Objective to obtain the recombinant GST protein (rSjGST), from Schistosoma japonicum and prepare its monoclonal antibody, and identify its biological characteristics, and establish a method for the detection of GST protein level in schistosomiasis japonicum patients. Methods the engineering strain BL21 containing recombinant plasmid pET28a-SjGST was cultured and induced by IPTG, then the Escherichia coli cells were broken and purified by His-tag affinity chromatography and high performance liquid chromatography (HPLC). The purified protein was identified by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. B lymphocyte hybridoma technique was used to immunize Balb/c female mice with high purity rSjGST protein. The positive clones were screened by indirect ELISA method and monoclonal cell lines were screened by limited dilution subcloning. Colchicine was used to identify its karyotype and ISO-2 mouse monoclonal antibody subtype detection kit was used to detect its subclass and its titer and specificity were detected. To establish a double antibody sandwich method and indirect ELISA method to detect serum GST levels, and to explore the precision, accuracy and specificity of the method. Results A large number of high purity rSjGST proteins were purified, and hybridoma cell lines 1B11 and 3C12, which could stably secrete anti rSjGST monoclonal antibodies, were screened. The subtypes of the McAb were identified as IgG2b and IgG1 by the kit. Conclusion the rSjGST protein was highly expressed by E. coli expression system. The monoclonal antibodies 1B11 and 3C12 were prepared by using this rSjGST protein. The double antibody sandwich ELISA method for detecting the level of serum rSjGST protein provided a basis for the future diagnosis of schistosomiasis. This diagnostic technique can also be used for serum detection of clinical schistosomiasis japonicum.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐宏波;程茂基;呂林;劉彬;羅緒剛;;飼糧添加有機(jī)鋅對(duì)斷奶仔豬飼喂效果的研究[J];黑龍江畜牧獸醫(yī);2009年05期

2 賈侃;趙r嚺，

本文編號(hào)：1866714